One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are tightly regulated and often overlap as evidenced by the existence of multiple protein kinase and phosphatase families and isoforms.
Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of these cascades. Consequently, considerable attention has been devoted to the characterization of proteins exhibiting either kinase or phosphatase enzymatic activity.
The magnitude and duration of signaling through mitogen-activated kinases (MAP kinases) and stress-activated kinases relay, amplify and integrate signals from a diverse range of stimuli and elicit an appropriate physiological response. In mammalian systems, these responses include cellular proliferation, differentiation, development, inflammatory responses and apoptosis (Keyse, Curr. Opin. Cell Biol., 2000, 12, 186-192).
Regulation of the signaling activities of MAP kinases and stress-activated kinases is dependent upon a balance between the activities of upstream activators and a complex network of protein phosphatases. These MAP kinase phosphatases include a class known as the dual specific (or dual specificity) phosphatases which share homology with a dual specificity phosphatase from vaccinia virus (VH-1) These dual specific phosphatase enzymes, which dephosphorylate tyrosine and threonine residues on the same substrate, can be divided into two groups, the inducible nuclear enzymes, and the cytosolic enzymes (Keyse, Curr. Opin. Cell Biol., 2000, 12, 186-192).
Dual specific phosphatase 5 (also known as hvH-3, B23 and DUSP-5) belongs to the inducible nuclear dual specific phosphatase group (Keyse, Curr. Opin. Cell Biol., 2000, 12, 186-192). It is expressed in a wide variety of tissues with relatively high expression in pancreas and brain (Ishibashi et al., J. Biol. Chem., 1994, 269, 29897-29902), as well as liver and placenta (Kwak and Dixon, J. Biol. Chem., 1995, 270, 1156-1160). It has been cloned (Ishibashi et al., J. Biol. Chem., 1994, 269, 29897-29902; Kwak and Dixon, J. Biol. Chem., 1995, 270, 1156-1160) and mapped to chromosome 10q25 (Martell et al., Genomics, 1994, 22, 462-464).
The primary biological function of dual specific phosphatase 5 is not yet confirmed. However, like other members of the inducible nuclear dual specific phosphatase group, it has been demonstrated to exhibit in vitro phosphatase activity toward extracellular signal regulated kinase-1 (ERK-1), a MAP kinase (Ishibashi et al., J. Biol. Chem., 1994, 269, 29897-29902). In addition, its expression is induced in human skin fibroblasts by heat shock and serum stimulation (Ishibashi et al., J. Biol. Chem., 1994, 269, 29897-29902) and in Reuber H4 cells in response to insulin and insulin growth factor-1 (Kwak and Dixon, J. Biol. Chem., 1995, 270, 1156-1160).
Rat1 fibroblasts transformed with the oncogenes Raf-1, v-Ha-Ras and v-src exhibit reduced levels of ERK-1 and ERK-2 activity. Normal levels of ERK-1 and ERK-2 activity in these cells were restored by treatment of the cells with vanadates which are non-specific inhibitors of dual specific phosphatases. This indicates that dysregulation of dual specific phosphatases may occur in transformed cells and affect the function of MAP kinases (Gopalbhai and Meloche, J. Cell Physiol., 1998, 174, 35-47).
The potential role of dual specific phosphatase 5 as a regulator of MAP kinase or stress-activated kinase activity indicates that its selective inhibition may be a potential strategy with which to derive treatments for hyperproliferative disorders, developmental disorders, inflammatory disorders and diseases arising from aberrant apoptosis.
Small molecules other than vanadates have been investigated as additional inhibitors of dual specific phosphatases. For example, Rice et al. have synthesized a library of dual specific phosphatase inhibitors based on a central oxazole pharmacophore (Rice et al., Biochemistry, 1997, 36, 15965-15974). To date however, there are no known examples of specific and selective inhibitors of dual specific phosphatase 5. Consequently, there remains a long felt need for agents capable of effectively and selectively inhibiting the function of dual specific phosphatase 5.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of dual specific phosphatase 5.
The present invention provides compositions and methods for modulating expression of dual specific phosphatase 5.